Ha probe y 11

Following transient transfection and/or drug treatment, whole-cell extracts (WCE) were prepared and immunoblots were performed as previously described [55 (link)]. The primary antibodies used to detect proteins were monoclonal anti-BRCA1 DO-9 (1: 200, Santa Cruz), monoclonal anti-HSP60 (1:1000, Sigma), monoclonal anti-HA (1:1000, Babco), polyclonal anti-pBRCA1Ser1497 (1:50, Upstate), polyclonal anti-γH2AX[pS139] (1:200, Cell Signaling Technology), monoclonal anti-Plk1 (1:500, Zymed), monoclonal anti-Myc epitope sequence (1:1000, Calbiochem) and polyclonal anti-Kap1 (1:2000; Abcam). The secondary antibodies were peroxidase-conjugated IgG (1:5000, Cell Signaling). Immunoblot signals were detected by using ECL (Pierce). For immunoprecipitation, 500 μg to 1 mg of precleared WCE were incubated with 10 μg of rabbit polyclonal anti-BRCA1 antibody (BD Biosciences) or 2 μg of rabbit polyclonal HA-probe Y-11 (Santa Cruz) or with 5 μg of rabbit polyclonal anti-Plk1 antibody (Calbiochem) at 4°C overnight. Immune complexes were recovered with protein A-sepharose beads (Pharmacia) and washed three times using lysis buffer. Then beads were subjected to Western Blot analysis. Control immunoprecipitation was performed without antibody.

The in vivo proteolytic activity of AaRseP was analyzed using E. coli KK211 (ΔrseA, ΔrseP) cells (Kanehara et al., 2002 ▸ ) as described previously (Akiyama et al., 2015 ▸ ; Hizukuri et al., 2017 ▸ ). E. coli KK211 (ΔrseA, ΔrseP) cells harboring pYH124 [HA-MBP-RseA(LY1)148] were transformed with the pKK11 plasmid (EcRseP-His₆-Myc; Kanehara et al., 2001 ▸ ), pTM748 (AaRseP-His₈), pTWV228 or plasmids encoding their derivatives. M9 medium (without CaCl₂; Miller, 1972 ▸ ) supplemented with 20 µg ml⁻¹ of each of the 20 amino acids, 2 µg ml⁻¹ thiamine, 0.4% glucose, 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and 5 mM cAMP was inoculated with transformed E. coli KK211 cells and grown at 30°C for 3 h. Proteins were precipitated by trichloroacetic acid (TCA) treatment and separated by Laemmli SDS–PAGE. Immunoblots with anti-HA, anti-His or anti-SecB antibodies were visualized using a Lumino LAS 4000 mini image analyzer (Cytiva) with ECL Prime Western Blotting Detection Reagents (Cytiva). Rabbit polyclonal anti-HA [HA-probe (Y-11), Santa Cruz Biotechnology] and anti-SecB (Miyake et al., 2020 ▸ ) antibodies were used for immunoblotting. For the detection of His-tagged proteins, anti-His antibodies from the Penta-His HRP Conjugate Kit (Qiagen) were used.